Interneuron Origins in the Embryonic Porcine Medial Ganglionic Eminence

Research Articles: Development/Plasticity/Repair Interneuron origins in the embryonic porcine medial ganglionic eminence https://doi.org/10.1523/JNEUROSCI.2738-20.2021

Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.2738-20.2021 Received: 27 October 2020 Revised: 18 December 2020 Accepted: 8 January 2021

This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data.

Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published.

2 Interneuron origins in the embryonic porcine medial ganglionic eminence

4 Mariana L. Casalia1, Tina Li1, Harrison Ramsay1, Pablo J. Ross2, Mercedes F. Paredes3,4

5 & Scott C. Baraban3-5

7 1Department of Neurological Surgery, University of California, San Francisco, San

8 Francisco, CA

9 2Department of Animal Science, University of California, Davis, Davis, CA

10 3Department of Neurology, University of California, San Francisco, San Francisco, CA

11 4Weill Institute for Neurosciences, University of California, San Francisco, San Francisco,

12 CA

13 5Helen Wills Institute for Neuroscience, University of California, Berkeley, Berkeley, CA

15 Abbreviated title: Porcine Medial Ganglionic Eminence

17 Correspondence should be addressed to S.C.B. (email: [email protected])

19 Pallial (cortical and hippocampal) Interneurons contribute to the complexity of

20 neural circuits and maintenance of normal brain function. Rodent interneurons

21 originate in embryonic ganglionic eminences, but developmental origins in other

22 species are less understood. Here, we show that transcription factor expression

23 patterns in porcine embryonic subpallium are similar to rodents, delineating a

24 distinct medial ganglionic eminence (MGE) progenitor domain. On the basis of

25 Nkx2.1, Lhx6 and Dlx2 expression, in vitro differentiation into neurons expressing

26 GABA and robust migratory capacity in explant assays, we propose that cortical

27 and hippocampal interneurons originate from a porcine MGE region. Following

28 xenotransplantation into adult male and female rat hippocampus, we further

29 demonstrate that porcine MGE progenitors, like those from rodents, migrate and

30 differentiate into morphologically distinct interneurons expressing GABA. Our

31 findings reveal that basic rules for interneuron development are conserved across

32 species, and that porcine embryonic MGE progenitors could serve as a valuable

33 source for interneuron-based xenotransplantation therapies.

35 Significance Statement

36 Here we demonstrate that porcine MGE, like rodents, exhibit a distinct transcriptional and

37 interneuron-specific antibody profile, in vitro migratory capacity and are amenable to

38 xenotransplantation. This is the first comprehensive examination of embryonic

39 interneuron origins in the pig, and because a rich neurodevelopmental literature on

40 embryonic mouse MGE exists (with some additional characterizations in other species

41 like monkey and human) our work allows direct neurodevelopmental comparisons with

42 this literature.

44 Introduction

45 Excitatory glutamatergic neurons and inhibitory GABAergic interneurons represent the

46 two primary neuronal populations in mammalian brain. Cortical and hippocampal

47 inhibitory interneurons are a diverse subset of the total neuronal population, mediate

48 many critical brain functions, and arise from embryonic subpallium regions (Fishell, 2007;

49 Gelman et al., 2012; Kessaris et al., 2014). A wide spectrum of neurological pathologies

50 is associated with loss, or dysfunction, of interneurons including, but not limited to,

51 epilepsy, autism spectrum disorder, Alzheimer’s disease and schizophrenia (Marin, 2012;

52 Inan et al., 2013; Paterno et al., 2020). Studies over the past decade demonstrated that

53 transplantation of interneurons offer great promise for treatment of these disorders

54 (Baraban et al., 2009; Alvarez-Buylla et al., 2000; Bráz et al., 2012; Anderson and

55 Baraban, 2012; Hunt et al., 2013; Tong et al., 2014; Donegan et al., 2017; Juarez-Salinas

56 et al., 2019; Zhu et al., 2019). Interneuron transplantation studies are largely based on

57 harvesting progenitor cells from a medial ganglionic eminence (MGE) subregion. MGE

58 progenitors give rise to cortical GABAergic interneurons subtypes expressing

59 parvalbumin (PV)- and somatostatin (SOM) (Gelman et al., 2012; Hu et al., 2017; Pelkey

60 et al., 2017) along with hippocampal neurogliaform and Ivy cells expressing nitric oxide

61 synthase (nNOS) (Tricoire et al., 2010). These interneurons exhibit highly migratory

62 capabilities and unique functions. To date, the majority of successful MGE progenitor cell

63 transplantations showing efficacy in preclinical animal models harvest fresh murine

64 embryonic tissue. Human “MGE-like” embryonic or induced pluripotent stem cell derived

65 interneurons could offer an alternative, however, these neurons fail to (i) migrate

66 extensively or (ii) differentiate to cortical or hippocampal interneuron subtypes seen with

67 embryonic MGE progenitors, and (iii) exhibit protracted functional maturation (Nicholas et

68 al., 2013; Liu et al., 2013; Maroof et al., 2014). Human stem cells also present a risk of

69 tumorigenesis (Carpentino et al., 2008). Although embryonic allografted fetal human

70 tissue in Parkinson's disease patients ameliorated some symptoms of this disease in the

71 1990s (Björklund et al., 2003), practical and ethical problems using aborted human fetal

72 tissue have since led to an extended exploration for an alternative source(s) of suitable

73 fetal material for transplantation.

76 Most successfully applied to organs, xenotransplantation from pig-to-nonhuman primate

77 (or even pig-to-human) raise the exciting possibility that embryonic porcine tissue -

78 perhaps coupled with techniques to genetically modify a donor pig using transgenic or

79 CRISPR/Cas9 technologies (Cowan et al., 2019; Hryhorowicz et al., 2020) - could be a

80 viable source of transplantable interneurons. These efforts are supported by data showing

81 similarities at the gene and protein level between humans and pigs (Sjöstedt et al., 2020).

82 It is well established that murine MGE can be defined by transient anatomical landmarks

83 and expression of a unique set of transcription factors e.g., Nkx2.1, Dlx2 and Lhx6 (Corbin

84 et al., 2003; Du et al., 2008; Flames et al., 2007; Gelman et al., 2012). Analysis in human

85 and macaque (nonhuman Old World) monkey demonstrated a recapitulation of these

86 murine MGE transcription factor expression patterns (Hansen et al., 2013; Ma et al.,

87 2013). Tangential migration of interneurons from MGE-to-cortex or -hippocampus is also

88 believed to be highly conserved across species. Although some features of porcine

89 ganglionic eminences are known, this information is limited to lateral ganglionic eminence

90 (LGE) (Jacoby et al., 1999), a subpallial region that mainly generates olfactory bulb

91 interneurons and lacks tangential migratory properties in vitro or following transplantation

92 into postnatal brain.

94 Here we analyzed temporal and regional expression of MGE-specific transcription factors

95 in fetal porcine brain at embryonic days 30 and 35 (E35). Cultured porcine progenitors

96 express MGE-specific transcription factors, differentiate to GABAergic SOM+

97 interneurons and migrate extensively in vitro. Similar to human, embryonic pig ganglionic

98 eminence organization showed distinct organization of cells into doublecortin-positive

99 clusters, a pattern not seen in murine MGE. Xenotransplantation of E35 MGE progenitors

100 into adult rat hippocampus resulted in migration across all hippocampal sub-fields and

101 differentiation into GABAergic SOM+ interneurons up to 60 days after transplantation

102 (DAT). Overall, patterns of transcription factor expression, differentiation into distinct

103 interneuron subpopulations and tangential migration capacity all appear to be conserved

104 from rodents to pig MGE during evolution.

105

106 Materials and Methods

107

108 Animals. Donor porcine (Sus scrofa domesticus) embryos were procured from

109 Department of Animal Science, University of California, Davis (English Large white) or

110 National Swine Resource and Research Center (NSRRC:0016 GFP NT92; Whitworth et

111 al., 2009), University of Missouri. Adult male Sprague Dawley rats (Rattus norvegicus)

112 were purchased from Charles River Laboratories (strain code 400) and housed under a

113 standard 12 hr light/dark cycle with food and water provided ad libitum. A total of 133 rats

114 were used for xenotransplantation studies (79 female; 54 male); 75 rats received

115 intraperitoneal (i.p.) injection of an immunosuppression cocktail containing

116 methylprednisolone acetate (2 mg/kg), cyclosporine (20 mg/kg) and azathioprine (5

117 mg/kg) 3 times per week (Table 1). All protocols and procedures were approved by the

118 Institutional Animal Care and Use Committee (IACUC protocol #AN181524-02) and

119 adhered to University of California, San Francisco (UCSF; San Francisco, CA) Laboratory

120 Animal Resource Center and United States Public Health policy on Humane Care and

121 Use of Laboratory Animals guidelines.

122

123 Tissue collection and processing. Individual embryonic day 30 (E30) or E35 pig

124 embryos were maintained on ice in 50 ml Falcon conical centrifuge tubes (Fisher

125 Scientific, catalog #14-432-22) with DMEM/F12 media. Sterile techniques were used

126 during isolation and microdissection. Cell viability on freshly dissected brain tissue was

127 assessed using Trypan Blue (Sigma); only tissue with t 90% viability were processed for

128 cryopreservation.

129

130 Immunohistochemistry. Embryonic pig heads were removed under a stereomicroscope

131 (Leica MZ12(5)) and placed individually in a 50 ml Falcon tube containing 30 ml of 4%

132 paraformaldehyde (PFA; wt./vol) in phosphate buffered saline (PBS) at 4°C for 48 hr. Rats

133 were deeply anesthetized and perfused with 300 ml of cold 4% paraformaldehyde

134 solution. Brains were removed and post fixed ON with 4% PFA. Tissue was sequentially

135 dehydrated and cryoprotected in 10%, 20% and 30% sucrose (wt./vol) in PBS at 4°C until

136 sinking. Tissue was embedded in O.C.T. compound (Tissue-Tek #4583) and frozen on a

137 dry ice/ethanol slush and stored at -80°C for serial sectioning on a Leica CM1900

138 cryostat. Thin tissue sections (30 Pm for pig tissue; 25 Pm for rat tissue) were collected

139 onto glass slides (SuperFrost Plus, catalog #12-550-15) or free floating in

140 cryopreservation solution (3 vol. glycerol, 3 vol. ethylene glycol, 4 vol 1x 0.1% Sodium

141 Azide in PBS) and stored at -20°C. Tissue on SuperFrost slides were stained using TSA

142 amplification kit (Perkin Elmer; # NEL701A001KT, # NEL704A001KT and #

143 NEL705A001KT). Briefly, cryosections were left at ON at RT, baked for 15 min at 60°C,

144 fixed for 15 min with 4% PFA. Antigen retrieval was performed using nearly boiling 10

145 mM sodium citrate solution (5.95-6.05 pH range). Sections were allowed to cool down to

146 RT and tissue was delineated with pap-pen (Abcam, # ab2601). Free floating sections

147 were left at RT for 2 hr prior 3 x 15 min washes with PBS to remove cryopreservation

148 solution. Tissue was double stained with a primary antibody and detected with Alexa

149 secondary antibodies (Table 2). After testing different commercially available antibodies,

150 we failed to identify one that could reliably distinguish pig PV+, calbindin+, calretinin+,

151 reelin+ or VIP+ interneurons. Nuclei staining was done for 5 min as a final step (Sigma;

152 H33342). Briefly, sections were incubated with 0.3% triton-x 100 in PBS for 60 min at RT,

153 incubated in blocking solution (10% Donkey serum, 1% BSA, 0,1% Triton-X 100 in PBS)

154 for 60 min and incubated ON in primary antibody at 4q C. The next day sections were

155 washed 3 x 10 min in 0.05% Triton-X 100 in PBS, incubated for 120 min at RT in

156 secondary antibodies. Sections were mounted onto charged slides (Fisher Scientific,

157 Superfrost Plus) using Fluoromount-G solution (Southern Biotech #0100-01).

158

159 Image processing and cell quantification. Images were acquired from fluorescently

160 labeled sections using Keyence B7-X710, Leica TCS SP5 or Nikon Eclipse Ni-E confocal

161 microscopes. For whole-brain visualization, images were acquired using a 10x objective

162 in Tilescan mode on a Keyence microscope with Z-stack to focus automatic stitching of

163 arrayed tiles. Live imaging movies were acquired on a Leica TCS SP5 using 10x

164 objective, 1.5x optical zoom in time intervals of 20 min. All labeled cells were counted

165 using FIJI (http://imagej.net) by an investigator blind to the status of the experiment. Gray

166 level intensity was set using thresholded binary images in FIJI Watershed.

167

168 Experimental Design and Statistical Analysis

169 Cell culture. MGE was dissociated into single cells using neuronal isolation enzyme mix,

170 as described previously45. A 50 Pl drop of 10% FBS in NIM (DMEM Ff12, NEAA 1:100,N2

171 1x, Heparin 1:1000, B27 1x, EGF, FGF) containing 5,000 cells/cm2 was placed onto a

172 polyornithine/laminin coated coverslip in 24-well plate for 4 hr to allow initial attachment.

173 An additional 100 Pl of 10% FBS in NIM was added for overnight (ON) incubation and

174 further attachment of cells to the coverslip. Next morning, all media containing FBS was

175 removed and fresh NIM with no FBS with factors (BDNF, GDNF, IGF and cAMP 1:5000

176 vol/vol) was added to start the differentiation process. Cells were allowed to differentiate

177 for 14 days with media change every 48 hr. Factors were freshly added to the media.

178 After cells reached desired differentiation stage, they were fixed in 4% PFA for 5 min at

179 room temperature (RT) and stored in 0.1% sodium azide (wt/vol) in PBS for histology.

180

181 Xenotransplantation. MGE was identified by anatomical landmarks and freshly

182 dissected, as described (Hunt et al., 2013; Casalia et al., 2017) (Fig. 9). Tissue was then

183 cryopreserved in 10% DMSO in DMEM/F12 using 1.5 ml cryotubes and stored in a liquid

184 nitrogen tank between -210°C and -196°C. On the day of xenotransplantation, cryotubes

185 were quickly thawed under running warm water with gentle shaking. Neuronal Isolation

186 Enzyme mix (Pierce #88285E) was used for cell dissociation (30 min at 37°C). Cell

187 viability on freshly thawed cells was assessed using Trypan Blue; only MGE progenitor

188 cells with t 80% viability were processed for xenotransplantation. Rats were anesthetized

189 with 1.5% to 2.5% isoflurane in oxygen (vol./vol.) and secured in a stereotaxic frame (Kopf

190 model #900). In a series of pilot studies, several needle sizes and cell volumes were

191 tested (Fig. 10). A protocol using single cell suspension of 50,000 cells in 0.5 Pl of

192 DMEM/F12 loaded into a 32G blunt Hamilton syringe was chosen. Target coordinates for

193 stratum radiatum of hippocampal CA3 subfield were initially verified in a series of pilot

194 dye injections (n = 3) at the following stereotaxic coordinates relative to bregma: anterior-

195 posterior (AP) -3.4; medial-lateral (ML) ± 2.7; dorsal-ventral (DV) -3.4. Cell deposits were

196 made using an automatic microinjection pump (WPI Microinjection System UMP3) at a

197 rate of 5 nl/sec. The needle remained in place for 60 sec and then slowly withdrawn.

198 Following xenotransplantation, the incision was closed with Vetbond (3M #1469SB) and

199 rats were placed under a heat lamp until fully recovered. A successful porcine MGE

200 xenotransplant was defined post hoc using histological techniques. Namely, the

201 presence of at least 40 GFP+ cells in a minimum of 2 sections of the hippocampus (25

202 μm thick sections, spaced 300 μm apart).

203

204 Explant assay. MGE was dissected and further sub-dissected in 4 sections (anterior-

205 dorsal (AD)/ anterior-ventral (AV)/ posterior-dorsal (PD), posterior-ventral (PV), see

206 schematic figure 6). Each sub-section was submerged in a Matrigel drop (Corning, #

207 356237) containing 50% of cell culture media (Neurobasal, 1x amphotericin, 1x penicillin-

208 streptomycin, 1x glutaMAX) and placed onto a Matrigel coated coverslip of a 24-well plate

209 at 37° C under hypoxic conditions (5% CO2, 8% O2 balanced N2). Explants were allowed

210 to attach ON with addition of 10% FBS to cell culture media. Next morning media

211 containing FBS was removed and replaced with serum free media. Explants were kept in

212 vitro for 6 days with media change every 48 hr. At the end of the process each coverslip

213 was carefully washed 3 times with PBS and fixed for 15 min in 4% PFA in PBS solution

214 for histological analysis.

215

216 Slice culture. E35 brains were removed out of the head in cold DMEM F12 media. Tissue

217 was embedded in 3.5% low melting point agarose in 310 mOsm artificial cerebrospinal

218 fluid (ACSF ; 125 mM NaCl, 2.5 mM KCl,1 mM MgCl2, 2 mM CaCl2, 1.25 mM NaH2PO4,

219 25 mM glucose, bubbled with 95% O2 / 5% CO2) and sliced using a Leica VT1200S

220 vibratome at 300 Pm thickness. Sections were allowed to stabilize for 20 min in bubbling

221 ACSF on ice before transferred and suspended on 0.4 Pm Millicell-CM slice culture inserts

222 (Millipore). Sections were cultured for 6 days at 37°C in 5% CO2, 8% O2 and balanced

223 N2. For time-lapse imaging, an adenovirus (AV-CMV-GFP, 1 × 1010; Vector Biolabs) at

224 a dilution of 1:50–1:500 was applied to the slices, which were then cultured at 37°C, 5%

225 CO2, 8% O2. For time-lapse imaging, cultures were then transferred to an inverted Leica

226 TCS SP5 confocal microscope with an on-stage incubator streaming 5% CO2, 5% O2,

227 and balanced N2 into the chamber. Slices were imaged using a 10× air objective at 20-

228 minute intervals for 72 hours with repositioning of the z-stacks every 6-8 hr.

229

230

231 Statistics. Data is expressed as mean ± s.e.m. (standard error of the mean). Statistical

232 significance was assessed using one- or two-way ANOVA with Kruskal-Wallis’s multiple

233 comparisons. P-values less than < 0.05 were considered significant.

234

235 Results

236 Temporal and regional identification of porcine embryonic MGE

237 Across several species (Lavdas et al., 1999; Metin et al., 2007; Tanaka et al., 2011;

238 Gelman et al., 2012; Hansen et al., 2013; Ma et al., 2013; Hu et al., 2017), ganglionic

239 eminences (GE) consisting of extensive proliferative cell masses are visible as distinct

240 elevations adjacent to, and protruding into, lateral ventricular walls. Identification of

241 embryonic lateral and medial GE subregions is guided by transient developmental

242 appearance of anatomical landmarks (Brazel et al., 2003; Flames et al., 2007). In serial

243 sections, an expansion of porcine embryonic forebrain occurs between E30-E35 along

244 with formation of a clear morphological boundary e.g., a sulcus in the ventricular surface

245 separating LGE and MGE (Fig. 1). GE subregions can also be identified based on

246 temporal transcription factor expression patterns. Nkx2.1, a homeobox transcription

247 factor gene, is required for MGE specification and highly expressed in early MGE

248 ventricular zones (Corbin et al., 2003; Du et al., 2008; Sandberg et al., 2016). At E30 and

249 E35, we observed prominent anterior-to-posterior Nkx2.1 expression coinciding with

250 appearance of a sulcus and anatomical designation of an MGE subregion (Fig. 1a,

251 arrows); absence of expression in LGE and CGE was also noted (Fig. 1b). Gsh2 (also

252 known as Gsx2), another homeobox transcription factor gene, is critical for dorsal-ventral

253 patterning of telencephalon and diffusely expressed throughout GE domains (Flames et

254 al., 2007; Du et al., 2008). Coronal sections at E30 show diffuse expression of Gsh2

255 throughout porcine embryonic GE while evolving to more restricted expression patterns

256 corresponding to CGE and LGE ventricular proliferative zone regions at E35 (Figs. 1a-b,

257 white arrows). Olig2, a basic helix-loop-helix transcription factor gene, is expressed in

258 progenitor cells of ventral GE with highest levels in Nkx2.1-expressing MGE subregions

259 (Miyoshi et al., 2007). At E35, Olig2 expression is prominent in proliferative zones of

260 porcine MGE with only sparse expression in CGE or LGE (Fig. 1b). Dlx homeobox

261 transcription factors (Dlx1/2 and Dlx5/6), widely expressed throughout subpallium, are

262 broadly required for GE progenitors to migrate and differentiate into GABAergic

263 interneurons (Flames et al., 2007; Du et al., 2008; Laclef and Metin, 2018). Porcine Dlx2

264 expression at E35 is prominent in MGE (and CGE) though somewhat less dense along

265 ventricular proliferative zones. LGE expression is more diffuse presumably because

266 interneuron progenitors begin migration toward cortical areas at this stage of embryonic

267 development. Optical density measurements on coronal sections confirm high levels of

268 Dlx2 and Olig2 expression in all three ventricular zones of porcine MGE at E30 and E35

269 (Fig. 1c). Lhx6, a LIM homeodomain transcription factor, activated by Nkx2.1 also plays

270 a critical role in specification of MGE-derived interneuron subtypes (Grigoriou et al., 1998;

271 Du et al., 2008). Lhx6, expressed in MGE subventricular and submantle zones, is required

272 for differentiation of PV+ and SOM+ interneurons36,37. At E30, we observed significant

273 Lhx6 expression as a column of migrating cells bordering ventricular zones, contiguous

274 with basal MGE and beginning to populate piriform cortex (pCx) ventrally and LGE

275 laterally (Fig. 2a, coronal and sagittal). Dlx2 shows a similar co-expression pattern but

276 overall density is less in piriform cortex and scattered expression in ventricular zones was

277 noted. At E35, Lhx6 expression in presumably migratory cells outside the MGE

278 ventricular zone is more prominent ventrally (Fig. 2a, coronal) along with scattered

279 expression into intermediate zones of LGE and cortex (Fig. 2a, coronal and sagittal; Fig.

280 3b). Dlx2 expression at E35 is prominent in MGE and LGE subregions. Nkx2.1 co-

281 expression with Lhx6 or Dlx2 can be seen in most cells within the MGE ventricular

282 proliferative zone at E35 (Fig. 2b1, coronal; Fig. 3a) but only a few cells in more

283 intermediate zones (Fig. 2b2, sagittal). Serial coronal sections along the anterior-to-

284 posterior axis (Fig. 2c) reveal distinct Lhx6 expression in presumed tangential

285 telencephalon-to-cortex, telencephalon-to-striatum, telencephalon-to-olfactory bulb

286 migratory streams, as previously described in mice (Marin and Rubenstein, 2001; Alifragis

287 et al., 2004; Liodis et al., 2007); Lhx6 co-expression with Dlx2 was also noted in MGE

288 (Fig. 3a). In medial coronal sections at E35, MGE (marked by intense Nkx2.1 and Lhx6

289 expression) co-expressed Dlx2 and Olig2 more densely than LGE (Figs. 3a-b).

290 Expression of CouptfII (an orphan nuclear receptor enriched in CGE)(Kanatani et al.,

291 2008) was expressed in dorsal MGE, as expected (Cai et al., 2013); Gsh2 (enriched in

292 LGE/CGE)(Du et al., 2008; Laclef and Metin, 2018) was not prominent in MGE (Fig. 3b).

293 Non-specific ventral forebrain progenitor markers (Ki67, Olig2 and DCX) label porcine

294 MGE at E35 with dense co-expression of Olig2 and Ki67 noted (Fig. 3b).

295

296 Next, we performed a compartmentalized quantitative analysis of Nxk2.1 and Dlx2

297 expressing cells using coronal sections representative of anterior or posterior levels of

298 E35 porcine MGE. MGE and LGE were divided into 3 subregions starting at the

299 ventricular surface (MGE1) as well as boundary regions for MGE/LGE or LGE/Cortex

300 (Fig. 3c). In the most anterior section (Fig. 3c, PH5 #127-1), Nkx2.1 cell density is highest

301 in MGE regions 1 and 2 corresponding to proliferative ventricular zones, and extending

302 into subregion 3 of the most posterior section (Fig. 3c, PH5 #137-1). At a posterior level,

303 Nkx2.1 cell density drops off at the MGE/LGE boundary and is absent from LGE and

304 LGE/Cortex boundaries. Dlx2 cell density is high throughout all MGE proliferative zones

305 and subregions, MGE/LGE boundary and into LGE subregions 1 and 2 (Fig. 3c). As

306 expected (Flames et al., 2007; Long et al., 2009), co-expression of cells expressing

307 Nkx2.1 and Dlx2 cells was restricted to MGE at both levels.

308

309 It is well established that MGE produces GABAergic neurons that tangentially migrate to

310 hippocampus, cortex, striatum and thalamus (Marin and Rubenstein, 2001; Metin et al.,

311 2007; Laclef and Metin, 2018). Next, we used down-regulation of Nkx2.1 expression in

312 migratory cells to delineate MGE regions coupled with Lhx6 expression to track migratory

313 cells and the transition between germinal zone and migratory routes at E35 (Fig. 4). Large

314 populations of Nkx2.1+ progenitor cells were observed in MGE with additional Nkx2.1+

315 cells migrating into caudate-striatum and thalamus. Nkx2.1+ cell density greatly

316 decreased in LGE consistent with down-regulation of this transcription factor after leaving

317 MGE (Marin and Rubenstein, 2001); Nkx2.1+ cells were not observed in cortex or

318 hippocampus. A significant population of Lhx6+ cells can be seen in dorsal MGE along

319 with CouptfII+ cells (Fig. 4). Outside MGE, similar patterns of Lhx6+ and CouptfII+ cells can

320 be seen in caudate-striatum, thalamus, and cortex suggesting a corridor for tangential

321 migration. Distinct non-overlapping populations of Nkx2.1+ and Lhx6+/CouptfII+ cells are

322 present in caudate-putamen and thalamus. Taken together, these observations support

323 a conclusion that temporal, morphological and molecular characteristics of embryonic

324 MGE are conserved in the pig.

325

326 At E35, we also noted a unique organization of DCX+ cells originating close to the ventricle

327 in VZ or SVZ regions of porcine MGE (Figs. 5a-b, inset #1). Organization of MGE

328 progenitor cells into clusters was described for human and primate (but not murine)

329 embryonic brains (Hansen et al., 2013; Ma et al., 2013). Some co-expression with a

330 marker of young proliferative Ki67+ cells was observed in these DCX+ cluster-like

331 formations. These cells extended transversally towards outer ventricular areas with co-

332 expression of Psa-NCAM, a neural cell adhesion molecule regulating maturation of

333 GABAergic interneurons42 (Figs. 5a-b, inset #2). More distinct oval-shaped clusters were

334 seen in outer VZ and SVZ further from the ventricular surface (Figs. 5a, c, insets #3-5).

335 Oval-shaped clusters were densely populated by a core of DCX+/Ki67+proliferative young

336 neurons and clear co-expression of Olig2 and Psa-NCAM.

337

338 In vitro migration and characterization of porcine MGE

339 Embryonic MGE progenitor cells exhibit chain migration when cultured in a three-

340 dimensional extracellular matrix gel (Matrigel)(Wichterle et al., 1997, 2003). Using the

341 same explant protocol, E35 porcine MGE sections were placed in Matrigel drops and kept

342 under hypoxic conditions for 6 days in vitro (6 DIV). Extensive migration from MGE

343 explants, forming an outer ring of cells emerging from the core, was seen as early as 3

344 DIV, and grew more extensive by 6 DIV (Fig. 6a, yellow arrow). Outside this band cells

345 were loosely arranged forming a meshwork of migrating cell chains emanating from the

346 explant core in all directions (Fig. 6a, magenta arrow) and reaching distant areas of the

347 plate (Fig. 6a, magenta arrowhead). Cells migrating outward from explants co-expressed:

348 (i) DCX, a microtubule-associated protein expressed by neuronal progenitor cells and

349 immature neurons and (ii) Lhx6, an MGE-specific transcription factor (Fig. 6a, middle and

350 lower panels). DCX+ cells had an elongated morphology resembling those described for

351 migrating mouse MGE cells in vitro (Wichterle et al., 1997). We also observed robust

352 individual cell migration in time-lapse movies obtained from E35 porcine MGE tissue

353 slices cultured in vitro for 48 hours (Supplemental Movie). To quantify migration capacity,

354 MGE explants were sub-divided into Anterior or Posterior and Dorsal or Ventral regions

355 (AD/PD/AV/PV) and individual cell distance from explants was measured (Fig. 6b,

356 schematic). As expected (Wichterle et al., 1997, 2003), migration was robust in every

357 sub-region (Fig. 6b).

358

359 In vitro differentiation of MGE progenitor cells

360 Under appropriate induction protocols (Franchi et al., 2018), MGE progenitors

361 differentiate into cortical or hippocampal GABAergic neurons in vitro. Therefore, porcine

362 MGE cells were cultured in vitro to evaluate molecular maturation. Cells dissected from

363 E35 porcine MGE were plated onto polyornithine laminin coated dishes and cultured as

364 monolayers. Immunohistochemical analysis was performed on day 14 (Fig. 7a). Many

365 cells with mature neuronal morphologies and extensive processes co-expressed

366 neurofilament (NF70) and neuron-specific class III β-tubulin (e.g., antigen for the Tuj1

367 antibody), a marker of immature and mature neurons. MGE-derived cells with complex

368 neuronal morphologies expressed markers of inhibitory interneurons, namely GABA and

369 a vesicular GABA transporter (VGAT). Consistent with an MGE identity, cells also

370 expressed OLIG2 (20.5%), LHX6 (35.8%) and SOM (12.8%) (Figs. 7a-b). Many cells

371 expressed DCX, a marker for young migratory neurons (Figs. 7a-b). Cells with neuronal

372 morphologies also expressed synaptophysin (SYN), a synaptic vesicle protein and MAP2,

373 a dendritic marker. Only a very small number of MGE-derived cells expressed GFAP

374 (3.3%), an astrocytic marker. Undifferentiated stem cells were still present at this point in

375 culture as indicated by expression of Ki67 (35.3%), a proliferative cell marker. The

376 majority of interneuron-like GABA+ cells co-express VGAT (GABA:VGAT = 68%) or

377 neuron-specific class III β-tubulin (TUJ:VGAT = 89.6%); significant co-expression of DCX

378 and SYN was also noted (DCX:SYN = 73.3%)(Fig. 7c).

379

380 Xenotransplantation of pig MGE in adult rats

381 Using embryonic brain slices (Anderson et al., 1997; Marin et al., 2000), in utero fate

382 mapping (Wichterle et al., 2001), Matrigel explant assays (Wichterle et al., 1997, 2003),

383 and in vivo transplantation (Wichterle et al., 1999; Alvarez-Dolado et al., 2006; Hunt et

384 al., 2013), it is now well established that postnatal brain is permissive for migration and

385 integration of transplanted embryonic murine MGE progenitors. To establish an efficient

386 method for xenotransplantation and assessment of porcine MGE progenitors in a host

387 brain, MGE was dissected from transgenic E35 pig embryos expressing GFP52. GFP

388 expression was used to track migration and differentiation following transplantation into

389 hippocampus of recipient adult wild-type Sprague-Dawley rats (n = 75; Table 1). At 30

390 DAT, GFP+ cells dispersed into most hippocampal sub-regions in dorsal and ventral

391 directions with widespread distribution in dentate gyrus and CA1 (Fig. 8a-b). Distribution

392 of porcine MGE-derived cells in host hippocampus increased between 7 and 14 DAT,

393 reaching a relative plateau up to 60 DAT (Fig. 8c). Porcine MGE-derived GFP+ cells in

394 hippocampus differentiated into cells resembling mature interneuron morphologies (e.g.,

395 bipolar cells, basket cells, and multi-polar cells) with elaborate dendritic trees and axonal

396 projections (Figs. 8b, 8d). None of the porcine MGE-derived neurons exhibited

397 morphological features of cortical pyramidal neurons (e.g., triangular cell soma extending

398 a thick spiny apical dendrite). No tumors were observed in any host animal.

399

400 MGE-derived interneurons can be identified by expression of the neurotransmitter GABA

401 and neuropeptides such as SOM or PV (Fishell, 2008; Gelman et al., 2012; Laclef and

402 Metin, 2018). Double-immunofluorescence revealed that most GFP+ porcine MGE-

403 derived cells express GABA between 14 and 60 DAT. GFP+ cells exhibited increased

404 subtype-specific SOM co-expression that also correlated with time post transplantation.

405 By 60 DAT, the percentage of SOM:GFP+ cells significantly increased to 26.6% (Fig. 11).

406 PV+ pMGE-derived neurons in the recipient rat brain could not be detected with available

407 antibodies (see Methods). Tuj1 was present in GFP+ cells at 30 and 60 DAT. GFP+ cells

408 expressing Olig2 (20.5%) were primarily localized to injection sites and only a small sub-

409 population express GFAP (4.7%) (Fig. 12).

410

411 Discussion

412 Here, we present strong evidence that porcine progenitors in embryonic medial ganglionic

413 eminences express Nkx2.1, Lhx6, and Dlx2. In vitro, porcine MGE progenitor cells exhibit

414 robust migratory properties and differentiate to neurons expressing GABA, vGAT and

415 somatostatin. Following xenotransplantation into adult rat hippocampus, porcine MGE

416 progenitors migrate extensively and differentiate to neurons with mature morphologies

417 co-expressing GABA and somatostatin. Thus, porcine MGE progenitors may offer a

418 valuable source of new inhibitory neurons for the types of transplantation-based

419 therapeutic applications already suggested for mouse MGE progenitors e.g., epilepsy,

420 Alzheimer’s disease and traumatic brain injury (Baraban et al., 2009; Anderson and

421 Baraban, 2012; Hunt et al., 2013; Tong et al., 2014; Zhu et al., 2019).

422

423 The transcription factor profiles, migratory behavior (in vitro and in vivo) and differentiation

424 into inhibitory interneurons observed in our porcine studies is remarkably similar to that

425 described for mouse, monkey or human MGE. Seminal studies described a subpallial

426 origin of cortical and hippocampal interneurons in rodents (Anderson et al., 1997; Marin

427 et al., 2000; Pleasure et al., 2012), and a primary role of MGE in producing a

428 subpopulation of interneurons has been extensively reviewed (Wonders and Anderson,

429 2006; Fishell, 2007; Gelman et al., 2012). From this work several families of transcription

430 factors controlling subpallial embryonic development and fate of interneurons originating

431 in MGE are well established. These include Lhx6, Olig2, Dlx1/2 and Nkx2.1 in embryonic

432 subpallial MGE (Du et al., 2008; Laclef and Metin, 2018) and Lhx6 in migrating MGE-

433 derived cortical interneurons (Alifragis et al., 2004; Liodis et al., 2007; Vogt et al., 2014).

434 It has also been demonstrated, through comparative analyses of gene expression

435 patterns in chick, frog, zebrafish, lamprey, monkey and human, that basic developmental

436 organization of the embryonic subpallium and interneuron MGE origin is conserved

437 across species (Hauptmann and Gerster, 2000; Puelles et al., 2000; Brox et al., 2004;

438 Molnár et al., 2006; Cheung et al., 2007; Hansen et al., 2013; Pombal et al., 2011; Ma et

439 al., 2013; Pauly et al., 2014). Our observations are entirely consistent with these

440 descriptions.

441

442 In contrast to an earlier “failed” attempt to expand MGE-derived neurons in vitro from

443 primary cells (Chen et al., 2013), our studies more closely replicate those of Franchi et al.

444 (2018) showing that in vitro differentiation of rodent MGE progenitors into cortical or

445 hippocampal interneurons involves outgrowth and elaboration of dendritic and axonal

446 arbors (see Fig. 7). Similarly, most cells become GABAergic as indicated by co-

447 expression of the inhibitory neurotransmitter GABA and presynaptic marker vGAT. At 14

448 DIV, porcine MGE cells also mimic the dense network of MAP2-positive dendrites and

449 expression of a postsynaptic marker (synaptophysin) seen with rodent cultures. Unlike

450 murine MGE, porcine MGE exhibited unique organization of DCX+ cells. This feature of

451 porcine embryonic subpallium is reminiscent of human fetal brain sections (Hansen et al.,

452 2013) and suggests a conservation of neurodevelopmental programs between pig and

453 human not seen in rodents. It has been suggested that segregation into distinct cell

454 clusters facilitates tangential migration of interneuron progenitors in developing brain. A

455 process which, at the molecular level, may be regulated by expression of cell adhesion

456 molecules such as fibronectin leucine rich-repeat transmembrane protein (Seiradake et

457 al., 2014; del Toro et al., 2017) or transmembrane ephrin-B proteins (Zimmer et al., 2011;

458 Dimidschstein et al., 2013). As such, further experimental analysis of porcine embryonic

459 development could provide more direct insights into mechanisms underlying interneuron

460 migration in higher species.

461

462 Another key attribute widely attributed to MGE progenitors is a capacity for migration

463 (Wichterle et al., 1999). While most neuronal progenitors migrate through anatomically

464 discontinuous cell-poor and cell-rich environments using radial glial scaffolds, chain

465 migration allows migration of young GABAergic neurons through cell-dense regions

466 independent of radial glial. Based on this unique migration capacity, MGE progenitors

467 transplanted into cortex, hippocampus, striatum or even spinal cord, early in postnatal

468 development or adult brain, retain a migratory capacity (Wichterle et al., 1999; Alvarez-

469 Dolado et al., 2006; Baraban et al., 2009; Martinez-Cedeno et al., 2010; Braz et al., 2012;

470 Hunt et al., 2013; Casalia et al., 2017). Taking advantage of these properties, MGE

471 progenitor cell transplantation has shown remarkable therapeutic capacity in preclinical

472 animal models of epilepsy (Baraban et al., 2009; Hunt et al., 2013; Casalia et al., 2017),

473 traumatic brain injury (Zhu et al., 2014), Alzheimer’s disease (Tong et al., 2014),

474 Parkinson’s disease (Martinez-Cedeno et al., 2010), and neuropathic pain (Braz et al.,

475 2012; Juarez-Salinas et al., 2019). Here we used a xenotransplantation protocol to

476 demonstrate that porcine MGE progenitors maintain this migratory capacity following

477 transplantation into adult rat hippocampus. Robust migration was seen up to 60 DAT with

478 cells differentiating to mature interneurons with complex dendritic arborizations and

479 expression of antibodies marking MGE-specific inhibitory cell populations e.g., GABA and

480 somatostatin. Only a relatively small number of cells expressed markers for astrocytes

481 (GFAP) and unlike human ‘MGE-like’ stem cell derived interneurons (Carpentino et al.,

482 2008) we never observed tumors. Previous work transplanting mouse MGE progenitors

483 also consistently shows neurons expressing GABA and somatostatin (Alvarez-Dolado et

484 al., 2006; Baraban et al., 2009; Hunt et al., 2013; Tong et al., 2014; Casalia et al., 2017;

485 Zhu et al., 2019). While early postnatal MGE mouse transplant studies (and these vary

486 by recipient brain region) report parvalbumin-positive neurons also consistent with an

487 MGE lineage, PV+ cells take longer to develop and make up a smaller population of MGE-

488 derived neurons in adult intra-hippocampal transplant studies (Hunt et al., 2013; Casalia

489 et al., 2017). We also noted that unlike same species allograft murine-to-murine MGE

490 transplant studies where immunosuppression protocols were unnecessary, even with a

491 cocktail of immunosuppressants a pig-to-rat xenotransplantation protocol was only

492 successful in about half of the animals. Not unexpected, as similar xenotransplant studies

493 evaluating the fate of MGE-like human cells only utilized SCID immunodeficient mice as

494 host animals (Nicholas et al., 2013; Maroof et al., 2014). The myriad immunoreactive

495 processes that could take place, even in a relatively protected environment such as the

496 central nervous system, remain to be optimized for these types of xenotransplantation

497 studies. We anticipate that future studies will address these factors and perhaps utilize

498 CRISPR-based gene editing technologies (Cowan et al., 2019; Hryhorowicz et al., 2020)

499 to generate donor pig embryos ideally suited to this purpose.

500

501 Acknowledgements

502 We thank Rosalia Paterno, Colleen Carpenter and Joseane Righes Marafiga for technical

503 support and advice. We also acknowledge the National Swine Resource Center support

504 in supplying pig embryos for our research under grant #U42-OD011140 (to NSRRC).

505 This work was supported by an NIH R37 award (NS071785) from the NINDS and a UCSF

506 Program for Breakthrough Biomedical Research award (to S.C.B.)

507

508 Author contributions

509 M.F.C., M.L.P. and S.C.B. designed experiments. M.F.C., H.R., M.L.P. and T.L. carried

510 out experiments and analyzed data. P.J.R. provided pig embryos and expertise. M.F.C.

511 and S.C.B wrote the paper.

512

513 Competing interests

514 The authors declare no competing financial interests.

515

516 Data availability

517 The data that support the findings of this study are available from the corresponding

518 author upon reasonable request.

519

520

521 References

522 Alifragis P, Liapi A, Parnavelas JG (2004). Lhx6 regulates the migration of cortical

523 interneurons from the ventral telencephalon but does not specify their GABA phenotype.

524 J Neurosci 24:5643-5648.

525 Alvarez-Buylla A, Herrera DG, Wichterle H (2000). The subventricular zone: source of

526 neuronal precursors for brain repair. Prog Brain Res 127:1-11.

527 Alvarez-Dolado M, Calcagnotto ME, Karkar KM, Southwell DG, Jones-Davis DM, Estrada

528 RC, Rubenstein JL, Alvarez-Buylla A, Baraban SC (2006). Cortical inhibition modified by

529 embryonic neural precursors grafted into the postnatal brain. J Neurosci 26:7380-7389.

530 Anderson SA, Baraban SC (2012). Cell Therapy Using GABAergic Neural Progenitors.

531 Jasper's Basic Mechanisms of the Epilepsies. (Noebels JL, Avoli M et al., ed) Bethesda

532 (MD).

533 Anderson SA, Eisenstat DD, Shi L, Rubenstein JL (1997). Interneuron migration from

534 basal forebrain to neocortex: dependence on Dlx genes. Science 278:474-476.

535 Baraban SC, Southwell DG, Estrada RC, Jones DL, Sebe JY, Alfaro-Cervello C, Garcia-

536 Verdugo JM, Rubenstein JL, Alvarez-Buylla A (2009). Reduction of seizures by

537 transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice.

538 Proc Natl Acad Sci U S A 106:15472-15477.

539 Bjorklund A, Dunnett SB, Brundin P, Stoessl AJ, Freed CR, Breeze RE, Levivier M,

540 Peschanski M, Studer L, Barker R (2003). Neural transplantation for the treatment of

541 Parkinson's disease. Lancet Neurol 2:437-445.

542 Braz JM, Sharif-Naeini R, Vogt D, Kriegstein A, Alvarez-Buylla A, Rubenstein JL,

543 Basbaum AI (2012). Forebrain GABAergic neuron precursors integrate into adult spinal

544 cord and reduce injury-induced neuropathic pain. Neuron 74:663-675.

545 Brazel CY, Romanko MJ, Rothstein RP, Levison SW (2003). Roles of the mammalian

546 subventricular zone in brain development. Prog Neurobiol 69:49-69.

547 Brox A, Puelles L, Ferreiro B, Medina L (2004). Expression of the genes Emx1, Tbr1, and

548 Eomes (Tbr2) in the telencephalon of Xenopus laevis confirms the existence of a ventral

549 pallial division in all tetrapods. J Comp Neurol 474:562-577.

550 Carpentino JE, Hartman NW, Grabel LB, Naegele JR (2008). Region-specific

551 differentiation of embryonic stem cell-derived neural progenitor transplants into the adult

552 mouse hippocampus following seizures. J Neurosci Res 86:512-524.

553 Casalia ML, Howard MA, Baraban SC (2017). Persistent seizure control in epileptic mice

554 transplanted with gamma-aminobutyric acid progenitors. Ann Neurol 82:530-542.

555 Chen YJ, Vogt D, Wang Y, Visel A, Silberberg SN, Nicholas CR, Danjo T, Pollack JL,

556 Pennacchio LA, Anderson S, Sasai Y, Baraban SC, Kriegstein AR, Alvarez-Buylla A,

557 Rubenstein JL (2013). Use of "MGE enhancers" for labeling and selection of embryonic

558 stem cell-derived medial ganglionic eminence (MGE) progenitors and neurons. PLoS One

559 8:e61956.

560 Cheung AF, Pollen AA, Tavare A, DeProto J, Molnar Z (2007). Comparative aspects of

561 cortical neurogenesis in vertebrates. J Anat 211:164-176.

562 Corbin JG, Rutlin M, Gaiano N, Fishell G (2003). Combinatorial function of the

563 homeodomain proteins Nkx2.1 and Gsh2 in ventral telencephalic patterning.

564 Development 130:4895-4906.

565 Cowan PJ, Hawthorne WJ, Nottle MB (2019). Xenogeneic transplantation and tolerance

566 in the era of CRISPR-Cas9. Curr Opin Organ Transplant 24:5-11.

567 Cox ET, Brennaman LH, Gable KL, Hamer RM, Glantz LA, Lamantia AS, Lieberman JA,

568 Gilmore JH, Maness PF, Jarskog LF (2009). Developmental regulation of neural cell

569 adhesion molecule in human prefrontal cortex. Neuroscience 162:96-105.

570 Del Toro D, Ruff T, Cederfjall E, Villalba A, Seyit-Bremer G, Borrell V,Klein R (2017).

571 Regulation of Cerebral Cortex Folding by Controlling Neuronal Migration via FLRT

572 Adhesion Molecules. Cell 169:621-635.

573 Dimidschstein J, Passante L, Dufour A, van den Ameele J, Tiberi L, Hrechdakian T,

574 Adams R, Klein R, Lie DC, Jossin Y, Vanderhaeghen P(2013). Ephrin-B1 controls the

575 columnar distribution of cortical pyramidal neurons by restricting their tangential

576 migration. Neuron 79:1123-1135.

577 Donegan JJ, Tyson JA, Branch SY, Beckstead MJ, Anderson SA, Lodge DJ (2017). Stem

578 cell-derived interneuron transplants as a treatment for schizophrenia: preclinical

579 validation in a rodent model. Mol Psychiatry 22:1492-1501.

580 Du T, Xu Q, Ocbina PJ, Anderson SA (2008). NKX2.1 specifies cortical interneuron fate

581 by activating Lhx6. Development 135:1559-1567.

582 Fishell G (2007). Perspectives on the developmental origins of cortical interneuron

583 diversity. Novartis Found Symp 288: 21-35; discussion 35-44, 96-28.

584 Flames N, Pla R, Gelman DM, Rubenstein JL, Puelles L, Marin O (2007). Delineation of

585 multiple subpallial progenitor domains by the combinatorial expression of transcriptional

586 codes. J Neurosci 27:9682-9695.

587 Franchi SA, Macco R, Astro V, Tonoli D, Savino E, Valtorta F, Sala K, Botta M, de Curtis

588 I (2017). A Method to Culture GABAergic Interneurons Derived from the Medial

589 Ganglionic Eminence. Front Cell Neurosci 11:423.

590 Gelman DM, Marin O, Rubenstein JLR (2012). The Generation of Cortical Interneurons.

591 Jasper's Basic Mechanisms of the Epilepsies. Eds., J. L. Noebels, M. Avoli et al. Bethesda

592 (MD).

593 Grigoriou M, Tucker AS, Sharpe PT, Pachnis V (1998). Expression and regulation of Lhx6

594 and Lhx7, a novel subfamily of LIM homeodomain encoding genes, suggests a role in

595 mammalian head development. Development 125:2063-2074.

596 Hansen DV, Lui JH, Flandin P, Yoshikawa K, Rubenstein JL, lvarez-Buylla A, Kriegstein

597 AR (2013). Non-epithelial stem cells and cortical interneuron production in the human

598 ganglionic eminences. Nat Neurosci 16:1576-1587.

599 Hauptmann G, Gerster T (2000). Regulatory gene expression patterns reveal transverse

600 and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech Dev 91:105-118.

601 Hryhorowicz M, Lipinski D, Hryhorowicz S, Nowak-Terpilowska A, Ryczek N, Zeyland J

602 (2020). Application of Genetically Engineered Pigs in Biomedical Research. Genes

603 (Basel) 11:670.

604 Hu JS, Vogt D, Sandberg M, Rubenstein JL (2017). Cortical interneuron development: a

605 tale of time and space. Development 144:3867-3878.

606 Hunt RF, Girskis KM, Rubenstein JL, Alvarez-Buylla A, Baraban SC (2013). GABA

607 progenitors grafted into the adult epileptic brain control seizures and abnormal behavior.

608 Nat Neurosci 16:692-697.

609 Inan M, Petros TJ, Anderson SA (2013). Losing your inhibition: linking cortical GABAergic

610 interneurons to schizophrenia. Neurobiol Dis 53:36-48.

611 Jacoby DB, Lindberg C, Cunningham MG, Ratliff J, Dinsmore J (1999). Long-term

612 survival of fetal porcine lateral ganglionic eminence cells in the hippocampus of rats. J

613 Neurosci Res 56:581-594.

614 Juarez-Salinas DL, Braz JM, Etlin A, Gee S, Sohal V, Basbaum AI (2019). GABAergic

615 cell transplants in the anterior cingulate cortex reduce neuropathic pain aversiveness.

616 Brain 142:2655-2669.

617 Kanatani S, Yozu M, Tabata H, Nakajima K (2008). COUP-TFII is preferentially expressed

618 in the caudal ganglionic eminence and is involved in the caudal migratory stream. J

619 Neurosci 28:13582-13591.

620 Kessaris N, Magno L, Rubin AN, Oliveira MG (2014). Genetic programs controlling

621 cortical interneuron fate. Curr Opin Neurobiol 26:79-87.

622 Laclef C, Metin C (2018). Conserved rules in embryonic development of cortical

623 interneurons. Semin Cell Dev Biol 76:86-100.

624 Lavdas AA, Grigoriou M, Pachnis V, Parnavelas JG (1999). The medial ganglionic

625 eminence gives rise to a population of early neurons in the developing cerebral cortex. J

626 Neurosci 19:7881-7888.

627 Liodis P, Denaxa M, Grigoriou M, Akufo-Addo C, Yanagawa Y, Pachnis V (2007). Lhx6

628 activity is required for the normal migration and specification of cortical interneuron

629 subtypes. J Neurosci 27:3078-3089.

630 Liu Y, Weick JP, Liu H, Krencik R, Zhang X, Ma L, Zhou GM, Ayala M, Zhang SC (2013).

631 Medial ganglionic eminence-like cells derived from human embryonic stem cells correct

632 learning and memory deficits. Nat Biotechnol 31:440-447.

633 Long JE, Cobos I, Potter GB, Rubenstein JL (2009). Dlx1&2 and Mash1 transcription

634 factors control MGE and CGE patterning and differentiation through parallel and

635 overlapping pathways. Cereb Cortex 19 Suppl 1:i96-106.

636 Ma T, Wang C, Wang L, Zhou X, Tian M, Zhang Q, Zhang Y, Li J, Liu Z, Cai Y, Liu F, You

637 Y, Chen C, Campbell K, Song H, Ma L, Rubenstein JL, Yang Z (2013). Subcortical origins

638 of human and monkey neocortical interneurons. Nat Neurosci 16:1588-1597.

639 Marin O (2012). "Interneuron dysfunction in psychiatric disorders. Nat Rev Neurosci

640 13:107-120.

641 Marin O, Anderson SA, Rubenstein JL (2000). Origin and molecular specification of

642 striatal interneurons. J Neurosci 20:6063-6076.

643 Marin O, Rubenstein JL (2001). A long, remarkable journey: tangential migration in the

644 telencephalon. Nat Rev Neurosci 2:780-790.

645 Maroof AM, Keros S, Tyson JA, Ying SW, Ganat YM, Merkle FT, Liu B, Goulburn A,

646 Stanley EG, Elefanty AG, Widmer HR, Eggan K, Goldstein PA, Anderson SA, Studer L

647 (2013). Directed differentiation and functional maturation of cortical interneurons from

648 human embryonic stem cells. Cell Stem Cell 12:559-572.

649 Martinez-Cerdeno V, Noctor SC, Espinosa A, Ariza J, Parker P, Orasji S, Daadi MM,

650 Bankiewicz K, Alvarez-Buylla A, Kriegstein AR (2010). Embryonic MGE precursor cells

651 grafted into adult rat striatum integrate and ameliorate motor symptoms in 6-OHDA-

652 lesioned rats. Cell Stem Cell 6:238-250.

653 Metin C, Alvarez C, Moudoux D, Vitalis T, Pieau C, Molnar Z (2007). Conserved pattern

654 of tangential neuronal migration during forebrain development. Development 134:2815-

655 2827.

656 Miyoshi G, Butt SJ, Takebayashi H, Fishell G (2007). Physiologically distinct temporal

657 cohorts of cortical interneurons arise from telencephalic Olig2-expressing precursors. J

658 Neurosci 27:7786-7798.

659 Molnar Z, Metin C, Stoykova A, Tarabykin V, Price DJ, Francis F, Meyer G, Dehay C,

660 Kennedy H (2006). Comparative aspects of cerebral cortical development. Eur J Neurosci

661 23:921-934.

662 Nicholas CR, Chen J, Tang Y, Southwell DG, Chalmers N, Vogt D, Arnold CM, Chen YJ,

663 Stanley EG, Elefanty AG, Sasai Y, Alvarez-Buylla A, Rubenstein JL, Kriegstein AR

664 (2013). Functional maturation of hPSC-derived forebrain interneurons requires an

665 extended timeline and mimics human neural development. Cell Stem Cell 12:573-586.

666 Paterno R, Casalia M, Baraban SC (2020). Interneuron deficits in neurodevelopmental

667 disorders: Implications for disease pathology and interneuron-based therapies. Eur J

668 Paediatr Neurol 24:81-88.

669 Pauly MC, Dobrossy MD, Nikkhah G, Winkler C, Piroth T(2013). Organization of the

670 human fetal subpallium. Front Neuroanat 7:54.

671 Pelkey KA, Chittajallu R, Craig MT, Tricoire L, Wester JC, McBain CJ (2017).

672 Hippocampal GABAergic Inhibitory Interneurons. Physiol Rev 97:1619-1747.

673 Pleasure SJ, Anderson S, Hevner R, Bagri A, Marin O, Lowenstein DH, Rubenstein JL

674 (2000). Cell migration from the ganglionic eminences is required for the development of

675 hippocampal GABAergic interneurons. Neuron 28:727-740.

676 Pombal MA, Alvarez-Otero R, Perez-Fernandez J, Solveira C, Megias M (2011).

677 Development and organization of the lamprey telencephalon with special reference to the

678 GABAergic system. Front Neuroanat 5:20.

679 Puelles L, Kuwana E, Puelles E, Bulfone A, Shimamura K, Keleher J, Smiga S,

680 Rubenstein JL (2000). Pallial and subpallial derivatives in the embryonic chick and mouse

681 telencephalon, traced by the expression of the genes Dlx-2, Emx-1, Nkx-2.1, Pax-6, and

682 Tbr-1. J Comp Neurol 424:409-438.

683 Sandberg M, Flandin P, Silberberg S, Su-Feher L, Price JD, Hu JS, Kim C, Visel A, Nord

684 AS, Rubenstein JLR (2016). Transcriptional Networks Controlled by NKX2-1 in the

685 Development of Forebrain GABAergic Neurons. Neuron 91:1260-1275.

686 Seiradake E, del Toro D, Nagel D, Cop F, Hartl R, Ruff T, Seyit-Bremer G, Harlos K,

687 Border EC, Acker-Palmer A, Jones EY, Klein R (2014). FLRT structure: balancing

688 repulsion and cell adhesion in cortical and vascular development. Neuron 84:370-385.

689 Sjostedt E, Zhong W, Fagerberg L, Karlsson M, Mitsios N, Adori C, Oksvold P, Edfors F,

690 Limiszewska A, Hikmet F, Huang J, Du Y, Lin L, Dong Z, Yang L, Liu X, Jiang H, Xu X,

691 Wang J, Yang H, Bolund L, Mardinoglu A, Zhang C, von Feilitzen K, Lindskog C, Ponten

692 F, Luo Y, Hokfelt T, Uhlen M, Mulder J (2020). An atlas of the protein-coding genes in the

693 human, pig, and mouse brain. Science 367:eaay5947.

694 Tanaka DH, Oiwa R, Sasaki E, Nakajima K (2011). Changes in cortical interneuron

695 migration contribute to the evolution of the neocortex. Proc Natl Acad Sci U S A 108:8015-

696 8020.

697 Tong LM, Djukic B, Arnold C, Gillespie AK, Yoon SY, Wang MM, Zhang O, Knoferle J,

698 Rubenstein JL, Alvarez-Buylla A, Huang Y (2014). Inhibitory interneuron progenitor

699 transplantation restores normal learning and memory in ApoE4 knock-in mice without or

700 with a-beta accumulation. J Neurosci 34:9506-9515.

701 Vogt D, Hunt RF, Mandal S, Sandberg M, Silberberg SN, Nagasawa T, Yang Z, Baraban

702 SC, Rubenstein JL (2014). Lhx6 directly regulates Arx and CXCR7 to determine cortical

703 interneuron fate and laminar position. Neuron 82:350-364.

704 Whitworth KM, Li R, Spate LD, Wax DM, Rieke A, Whyte JJ, Manandhar G, Sutovsky M,

705 Green JA, Sutovsky P, Prather RS (2009). Method of oocyte activation affects cloning

706 efficiency in pigs. Mol Reprod Dev 76:490-500.

707 Wichterle H, Alvarez-Dolado M, Erskine L, Alvarez-Buylla A (2003). Permissive corridor

708 and diffusible gradients direct medial ganglionic eminence cell migration to the neocortex.

709 Proc Natl Acad Sci U S A 100:727-732.

710 Wichterle H, Garcia-Verdugo JM, Alvarez-Buylla A (1997). Direct evidence for homotypic,

711 glia-independent neuronal migration. Neuron 18:779-791.

712 Wichterle H, Garcia-Verdugo JM, Herrera DG, Alvarez-Buylla A (1999). Young neurons

713 from medial ganglionic eminence disperse in adult and embryonic brain. Nat Neurosci

714 2:461-466.

715 Wichterle H, Turnbull DH, Nery S, Fishell G, Alvarez-Buylla A (2001). "n utero fate

716 mapping reveals distinct migratory pathways and fates of neurons born in the mammalian

717 basal forebrain. Development 128:3759-3771.

718 Wonders CP, Anderson SA (2006). The origin and specification of cortical interneurons.

719 Nat Rev Neurosci 7:687-696.

720 Zimmer G, Rudolph J, Landmann J, Gerstmann K, Steinecke A, Gampe C, Bolz J (2011).

721 Bidirectional ephrinB3/EphA4 signaling mediates the segregation of medial ganglionic

722 eminence- and preoptic area-derived interneurons in the deep and superficial migratory

723 stream. J Neurosci 31:18364-18380.

724

725 726

727 Table 1: Porcine MGE donor cell manipulations

post hoc histology donor cells total (n) transplant survival rate (%) with cells no cells pMGE wt + lenti CMV GFP / immunosup 10 15 25 40 pMGE wt + lenti CMV GFP / NO immunosup 1 19 20 5 728 pMGE GFP / immunosup 19 31 50 38

729 *immunosuppression cocktail: methylprednisolone acetate (2 mg/kg), cyclosporine (20

730 mg/kg) and azathioprine (5 mg/kg)

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

747

748

749 Table 2: Antibodies

750

751

primary antibodies host specie company catalog # dilution CoupTFII mouse Perseus Proteomics PP-H7147-00 1/100 DBX1 rabbit abcam ab156283 1/500 DCX mouse Santa Cruz Biotechnology sc-271390 1/100 Dlx2 mouse Santa Cruz Biotechnology sc-393879 1/500 GABA rabbit sigma a2052 1/500 GFAP mouse EnCore MCA-5C10 1/1000 GFAP rabbit Dako Z0334 1/500 KI67 mouse BD pharmingen 556003 1/500 KI67 rabbit proteintech 27309-1-AP 1/1000 LHX6 rabbit Santa Cruz Biotechnology sc-98607 1/100 LHX6 mouse Santa Cruz Biotechnology sc-271433 1/500 MAP2 mouse EnCore MCA-4h5 1/1000 Nkx2.1 (TTF1) mouse Progen 16108 1/20 Olig2 rabbit EMD Millipore AB9610 1/500 Pax6 mouse abcam ab78545 1/500 PSA-NCAM (IgM isotype) mouse millipore MAB5324 1/500 PV rabbit swant PV27 n/a PV rabbit abcam ab11427 n/a PV chicken EnCore CPCA-Pvalb n/a Somatostatin mouse Santa Cruz Biotechnology sc55565 1/500 Somatostatin goat Santa Cruz Biotechnology sc-7819 1/200 Synaptophysin mouse Santa Cruz Biotechnology sc17750 1/100 Tau chicken EnCore CPCA-Tau 1/500 Tuj (BIII tubulin) rabbit covance MRB-435P 1/1000 VGAT mouse Santa Cruz Biotechnology sc 393373 1/100 VIP Rabbit abcam AB22736 n/a nNOS rabbit millipore AB5380 n/a Calbindin rabbit Chemicon AB1778 n/a Calretinin mouse Chemicon MAB1568 n/a Calretinin rabbit Millipore AB5054 n/a Calretinin Mouse Swant 7697 n/a reelin mouse millipore MAB5364 n/a Reelin Mouse Calbiochem 553730 n/a Neuropeptide Y Rabbit Abcam ab30914 n/a amplification system for E35 stains TSA Biotin System PerkinElmer NEL700A001KT

752

753

754

755 Figure Legends

756 Figure 1: Developmental expansion of ganglionic eminences in the pig embryonic

757 brain. (a) Serial coronal sections of E30 and E35 pig brain along the anterior (A) to

758 posterior (P) axis. Sections labeled with antibodies recognizing regional transcription

759 factors that specify ganglionic eminences, Nkx2.1 (red) and Gsh2 (purple). DAPI cell

760 stain shown in blue. Arrows distinguish an anatomically defined MGE subregion; sulcus

761 establishing a boundary between LGE and MGE became apparent at E35 (yellow arrows)

762 and Gsh2 expression in LGE and CGE subventricular zone (white arrows) (E30, n = 9;

763 E35, n = 9). (b) Regional (Nkx2.1, Gsh2) and temporal (Olig2, Dlx2) transcription factor

764 expression at E35 for MGE, LGE, and CGE subregions (n = 9). Note distinct MGE-specific

765 Nkx2.1 expression. Scale bar = 100 Pm (c) Grouped summary data plots showing

766 intermediate progenitor (Dlx2) and undifferentiated radial glia (Olig2) expression at E30

767 and E35 (n = 3). Schematic (inset) shows where ventricular zone (VZ), subventricular

768 zone 1 (SVZ1) and 2 (SVZ2) optical density (O.D.) measurements were made.

769

770 Figure 2: MGE-specific transcription factor expression the developing porcine

771 ventral forebrain. (a) Representative coronal and sagittal sections containing the medial

772 GE region are shown at E30 and E35. Dlx2-expressing intermediate and Lhx6-expression

773 progenitors exit the ventricular zone (VZ) to begin a tangential migration toward cortical

774 destinations (n = 9). Scale bar = 500 Pm. (b) Higher magnification of inset 1 and 2 of

775 panel (a) showing MGE expression of Nkx2.1 expression. Note high co-expression of

776 Nxk2.1 and Lhx6 (left panels) or Dlx2 (right panels), in coronal and sagittal MGE sections.

777 Scale bar = 50 Pm (c) Serial coronal sections of E35 brain delineating Lhx6 expression

778 corresponding to MGE progenitor migratory routes along the anterior-to-posterior axis (n

779 = 9). Abbreviations: ventricular zone (VZ), medial ganglionic eminence (MGE), lateral

780 ganglionic eminence (LGE) and piriform cortex (pCX).

781

782 Figure 3: Demarcation of an embryonic MGE subregion in the developing pig brain.

783 (a) Co-expression of Nxk2.1, Dlx2 and Olig2 in a representative coronal brain section at

784 E35 (top panel). Higher resolution images shown in lower panels (box) highlight co-

785 expression patterns for intermediate progenitors (Nkx2.1 and Dlx2), MGE-specific

786 transcription factors (Lhx6 and Dlx2), proliferating and young neurons (Ki67 and DCX) or

787 MGE-derived oligodendrocytes and proliferating cells (Olig2 and Ki67) (n = 9). (b)

788 Schematic showing localization of MGE and LGE subregions on an equivalent section

789 corresponding to the gray scale images (top panel). Transcription factor markers (Lhx6

790 and Dlx2) label the porcine MGE (marked by Nkx2.1). Olig2 expression is also prominent

791 in MGE. CouptfII expression overlaps with migratory cells exiting the MGE. Gsh2

792 expression in VZ regions are most prominently in LGE (n = 18). (c) Representative

793 coronal sections (#127 & #137) along the Anterior-to-Posterior at 300 Pm are shown at

794 E35 brain. Nxk2.1+, Dlx2+ and Nkx2.1:Dlx2+ expressing cells were counted. MGE and

795 LGE subregions, designated numbers 1-3, were used for cell density measurements (in

796 cells/cm2). Heatmaps of these GE region cell density measurements are shown at left.

797 Heatmap color scale = 0 – 150,000 cells/cm2 (n = 18).

798

799 Figure 4: MGE-derived progenitors cell distribution in porcine embryonic

800 subpallium. (a) Representative Nkx2.1, CouptfII and Lhx6 expression patterns in porcine

801 at E35 are shown. DAPI stain and merged images are also shown (n = 9) (scale bar =

802 100 Pm). (b) Grouped summary data plots showing transcription factor (Nxk2.1, Lhx6

803 and CouptfII) cell densities (cell/cm2) for embryonic subregions shown in schematic (at

804 right). Co-expression quantification is also shown for Lhx6:CouptfII+, Nkx2.1:Lhx6+ and

805 Nkx2.1:CouptfII+ cells in these subregions (n = 7). Abbreviations: MGE, medial ganglionic

806 eminence; LGE, lateral ganglionic eminence; Cp-St, caudate putamen-Striatum; Cb,

807 cortical boundary; Cx, cortex; Hip, hippocampus; and Th, Thalamus.

808

809 Figure 5: Organization of DCX+ clusters in embryonic porcine MGE at E35. (a)

810 Representative coronal and sagittal sections showing DCX clustering in the MGE

811 (designated by white boundary). Note DCX+ or Psa-NCAM+ cell organization in panels at

812 left. (b) Magnified images of boxed areas in (a) show organization of cells co-expressing

813 Ki67 and Olig2 (#1) or DCX and Psa-NCAM (#2). (c) Magnified images of boxed areas in

814 (a) show clusters of cells co-expressing DCX, Ki67 and Olig2 (#3-4, subventricular and

815 ventricular zones, respectively) or DXC and Psa-NCAM (#5). Scale bar = 50 Pm. (n = 18)

816

817 Figure 6: Migration of porcine embryonic MGE progenitors in vitro. (a) Matrigel

818 explant assay showing migratory MGE progenitor cells (DAPI) immunolabeled with

819 antibodies recognizing DCX+ and Lhx6+ cells (top left, merged image; middle left,

820 individual images for DAPI, DCX and Lhx6; bottom left, higher magnification images for

821 individual cells labeled with DCX and Lhx6; merged image at far right). Differential

822 interference contrast bright-field image of embryonic explants at 4x magnification (top

823 right). A homogeneous wave of migratory cells emerging from the explant core in all

824 directions is marked in yellow; individual cells migrating to the outer reaches of the plate

825 are marked in magenta (n = 12). (b) Schematics of the dissection approach for explant

826 assay. Horizontal view of the GE (top left). MGE sub-dissected into 4 sections (AD,

827 anterior-dorsal; AV, anterior-ventral; PD, posterior-dorsal; PV, posterior-ventral; top

828 middle). Schematic representing from (a) showing distance measures from the explant

829 (yellow and magenta arrows)(top right). Bar plot of explant migration assays measured

830 as “distance from explant” (arbitrary unit) for all 4 MGE subregions (n = 4). Error bars

831 represent s.e.m.

832

833 Figure 7. Characterization of porcine MGE progenitor cells in vitro. (a)

834 Representative images of MGE-derived cells immunolabeled with markers of neuronal

835 maturation (NF70, neurofilament; TUJ, neuron-specific class III beta-tubulin) and DCX,

836 (doublecortin), interneurons (GABA, gamma-butyric acid; SOM, somatostatin or VGAT,

837 vesicular glutamate transporter), astrocytes (GFAP, glial fibrillary acidic protein) or

838 presynaptic terminals (SYN, synaptophysin). Images were collected at 14 days in culture.

839 (b) Quantification of expression plotted as a % over DAPI stain. Data are means r s.e.m.

840 (n = 3 or more independent experiments).

841

842 Figure 8. Distribution of porcine MGE-derived cells in the adult rat brain. (a)

843 Hippocampus of adult Sprague-Dawley rat recipients 60 DAT labeled for DAPI (blue) and

844 GFP+ transplanted porcine (p) MGE-derived neurons (green). MGE cells dispersed after

845 xenotransplantation into recipient rats populating dorsal and ventral hippocampus

846 locations. Higher magnification images show GFP+ cells in the rat hilus/dentate gyrus

847 (DG) and CA1 subregions. (b) GFP+ cell dispersion along the anterior-to-posterior axis of

848 the rat hippocampus at 7, 14, 30 and 60 DAT (n = 3 or more independent experiments).

849 (d) Porcine MGE-derived cells in the rat hippocampus differentiated into neurons

850 presenting typical morphology of interneuron subtypes e.g., bitufted, multipolar and

851 basket.

852

853 Figure 9. Dissection protocol for porcine MGE at E35. (a) Intact pig embryo. (b) Pig

854 head viewed from the top. (c) Isolated pig brain with first posterior cut to isolate forebrain

855 (d) Isolated pig brain micro-dissected into right and left hemispheres. Note: isolated right

856 hemisphere has GE exposed. (e) Higher magnification image to view exposed GE region

857 of right hemisphere.

858

859 Figure 10. Optimization of porcine MGE xenotransplant protocol. A range of needle

860 gauge sizes, injection volumes and cell densities were tested (n = 3 independent

861 experiments per manipulation). (a) 100,000 porcine MGE GFP+ (green) cells in 1 Pl

862 transplanted into adult rat hippocampus using a 26G blunt Hamilton needle. Note the

863 dense expression of GFAP-expressing MGE cells at 14 DAT (top panel). Higher

864 magnification to show GFP+ cells with astrocytic morphology (lower left panel). Also note

865 the high density of GFP:GFAP+ cells in fimbria and white matter tracts adjacent to

866 hippocampus (lower middle panel). The injection site also shows a dense concentration

867 of GFP+ cells unable to migrate out and astrocytes invading the needle tract (lower right

868 panel). (b) Plot showing quantification of GFP:GFAP+ cells in white matter tract (80.9%)

869 adjacent to hippocampus and inside hippocampus (55.9%). Mean r s.e.m. (c)

870 Representative images of the adult hippocampus (14 DAT) with GFP-labeled pMGE cells

871 transplanted at varying injection needle gauges, injection volumes and cell densities. (d)

872 Note the dense core of GFP+ cells near the injection site. Plot showing limited dispersion

873 of GFP:GFAP+ cells in hippocampus. Mean r s.e.m. (e) 50,000 porcine MGE GFP+

874 (green) cells in 0.5 Pl transplanted into adult rat hippocampus using a 32G blunt Hamilton

875 needle. Note the dispersion of GFP+ cells from the injection site and GFP:DCX+ neurons

876 at 14 DAT (f) Plot showing % of GFP+ cells co-expressing DCX (90.9%), GFAP (20.5%),

877 or Olig2 (12.1%).

878

879 Figure 11. Immunohistochemical characterization of pMGE-derived cells in the

880 adult rat brain. (a) Co-expression of GFP-labeled MGE-derived cells (green) with

881 markers for inhibitory interneurons (GABA and SOM) or a neuronal marker (Tuj). (b)

882 Quantification of pMGE-derived GFP+ cells co-expressing each marker at 14, 30 and 60

883 DAT. SOM 7 vs. 60 DAT: F(2,32) = 3.902; P = 0.0345, one-way ANOVA. Data are means

884 r s.e.m. (n = 3 or more independent experiments).

885

886 Figure 12. Immunohistochemical characterization of porcine MGE-derived cells in

887 the adult rat brain. (a) Co-expression of GFP-labeled MGE-derived cells (green) with

888 markers for astrocytes (GFAP) or oligodendrocytes (Olig2). (b) Quantification of MGE-

889 derived GFP+ cells co-expressing each marker at 14, 30 and 60 DAT. Data are means r

890 s.e.m. (n = 3 or more independent experiments).

891

892

893 Extended Data 894 Time-lapse movie of lentivirus-labeled pig MGE. Time-lapse movie showing migrating

895 (green) MGE neurons in a cultured embryonic pMGE tissue slice at E35 (n = 3

896 experiments).

897

898

899

900

901

902

903